Biogeosciences, 15, 137–157, 2018 https://doi.org/10.5194/bg-15-137-2018 © Author(s) 2018. This work is distributed under the Creative Commons Attribution 3.0 License.

Microbial methanogenesis in the -reducing zone of sediments in the Eckernförde Bay, SW Baltic Sea

Johanna Maltby1,a, Lea Steinle2,1, Carolin R. Löscher3,1, Hermann W. Bange1, Martin A. Fischer4, Mark Schmidt1, and Tina Treude5,6 1GEOMAR Helmholtz Centre for Ocean Research Kiel, Department of Marine Biogeochemistry, 24148 Kiel, Germany 2Department of Environmental Sciences, University of Basel, 4056 Basel, Switzerland 3Nordic Center for Earth Evolution, University of Southern Denmark, 5230 Odense, Denmark 4Institute of Microbiology, Christian-Albrecht-University Kiel, 24118 Kiel, Germany 5Department of Earth, Planetary, and Space Sciences, University of California Los Angeles (UCLA), Los Angeles, California 90095-1567, USA 6Department of Atmospheric and Oceanic Sciences, University of California Los Angeles (UCLA), Los Angeles, California 90095-1567, USA apresent address: Natural Sciences Department, Saint Joseph’s College, Standish, Maine 04084, USA

Correspondence: Johanna Maltby ([email protected]) and Tina Treude ([email protected])

Received: 4 February 2017 – Discussion started: 7 March 2017 Revised: 30 October 2017 – Accepted: 8 November 2017 – Published: 10 January 2018

Abstract. Benthic microbial methanogenesis is a known way of methanogenesis in the presence or absence of sul- source of in marine systems. In most sediments, fate reducers, which after the addition of a noncompetitive the majority of methanogenesis is located below the sulfate- substrate was studied in four experimental setups: (1) un- reducing zone, as sulfate reducers outcompete altered sediment batch incubations (net methanogenesis), for the major substrates and acetate. The coex- (2) 14C-bicarbonate labeling experiments (hydrogenotrophic istence of methanogenesis and sulfate reduction has been methanogenesis), (3) manipulated experiments with the ad- shown before and is possible through the usage of non- dition of either molybdate (sulfate reducer inhibitor), 2- competitive substrates by methanogens such as or bromoethanesulfonate ( inhibitor), or methanol methylated amines. However, knowledge about the magni- (noncompetitive substrate, potential methanogenesis), and tude, seasonality, and environmental controls of this noncom- (4) the addition of 13C-labeled methanol (potential methy- petitive methane production is sparse. In the present study, lotrophic methanogenesis). After incubation with methanol, the presence of methanogenesis within the sulfate reduc- molecular analyses were conducted to identify key functional tion zone (SRZ methanogenesis) was investigated in sed- methanogenic groups during methylotrophic methanogene- iments (0–30 cm below seafloor, cm b.s.f.) of the season- sis. To also compare the magnitudes of SRZ methanogen- ally hypoxic Eckernförde Bay in the southwestern Baltic esis with methanogenesis below the sulfate reduction zone Sea. Water column parameters such as , temperature, (> 30 cm b.s.f.), hydrogenotrophic methanogenesis was de- and salinity together with porewater geochemistry and ben- termined by 14C-bicarbonate radiotracer incubation in sam- thic methanogenesis rates were determined in the sampling ples collected in September 2013. area “Boknis Eck” quarterly from March 2013 to Septem- SRZ methanogenesis changed seasonally in the up- ber 2014 to investigate the effect of seasonal environmental per 30 cm b.s.f. with rates increasing from March changes on the rate and distribution of SRZ methanogene- (0.2 nmol cm−3 d−1) to November (1.3 nmol cm−3 d−1) sis, to estimate its potential contribution to benthic methane 2013 and March (0.2 nmol cm−3 d−1) to September emissions, and to identify the potential methanogenic groups (0.4 nmol cm−3 d−1) 2014. Its magnitude and distribution responsible for SRZ methanogenesis. The metabolic path- appeared to be controlled by organic matter availability,

Published by Copernicus Publications on behalf of the European Geosciences Union. 138 J. Maltby et al.: Microbial methanogenesis in the sulfate-reducing zone

C / N, temperature, and oxygen in the water column, In marine sediments, methanogenesis activity is mostly revealing higher rates in the warm, stratified, hypoxic restricted to the sediment layers below sulfate reduction seasons (September–November) compared to the colder, due to the successful competition of sulfate reducers with oxygenated seasons (March–June) of each year. The ma- methanogens for the mutual substrates acetate and hydrogen jority of SRZ methanogenesis was likely driven by the (H2; Oremland and Polcin, 1982; Crill and Martens, 1986; usage of noncompetitive substrates (e.g., methanol and Jørgensen, 2006). Methanogens produce methane mainly methylated compounds) to avoid competition with sulfate from using acetate (acetoclastic methanogenesis) or H2 and reducers, as was indicated by the 1000–3000-fold increase (CO2; hydrogenotrophic methanogenesis). in potential methanogenesis activity observed after methanol Competition with sulfate reducers can be relieved through addition. Accordingly, competitive hydrogenotrophic the usage of noncompetitive substrates (e.g., methanol or methanogenesis increased in the sediment only below methylated compounds, methylotrophic methanogenesis; Ci- the depth of sulfate penetration (> 30 cm b.s.f.). Members cerone and Oremland, 1988; Oremland and Polcin, 1982). of the family Methanosarcinaceae, which are known for The coexistence of sulfate reduction and methanogenesis has methylotrophic methanogenesis, were detected by PCR been detected in a few studies from organic-rich sediments, using Methanosarcinaceae-specific primers and are likely to e.g., salt-marsh sediments (Oremland et al., 1982; Buckley et be responsible for the observed SRZ methanogenesis. al., 2008), coastal sediments (Holmer and Kristensen, 1994; The present study indicates that SRZ methanogenesis is an Jørgensen and Parkes, 2010), or sediments in upwelling re- important component of the benthic methane budget and car- gions (Pimenov et al., 1993; Ferdelman et al., 1997; Maltby bon cycling in Eckernförde Bay. Although its contributions et al., 2016), indicating the importance of these environments to from the sediment into the water col- for methanogenesis within the sulfate reduction zone (SRZ umn are probably minor, SRZ methanogenesis could directly methanogenesis). So far, however, environmental controls of feed into methane oxidation above the sulfate–methane tran- SRZ methanogenesis remain elusive. sition zone. The coastal inlet Eckernförde Bay (southwestern Baltic Sea) is an excellent model environment to study seasonal and environmental controls of benthic SRZ methanogene- sis. Here, the muddy sediments are characterized by high 1 Introduction organic loading and high sedimentation rates (Whiticar, 2002), which lead to anoxic conditions within the upper- After water vapor and carbon dioxide, methane is the most most 0.1–0.2 cm b.s.f. (Preisler et al., 2007). Seasonally hy- abundant in the atmosphere (e.g., Hartmann poxic (dissolved oxygen < 63 µM) and anoxic (dissolved et al., 2013; Denman et al., 2007). Its atmospheric concen- oxygen = 0 µM) events in the bottom water of Eckernförde tration has increased more than 150 % since preindustrial Bay (Lennartz et al., 2014; Steinle et al., 2017) provide ideal times, mainly through increased human activities such as fos- conditions for anaerobic processes at the sediment surface. sil fuel usage and livestock breeding (Hartmann et al., 2013; Sulfate reduction is the dominant pathway of organic car- Wuebbles and Hayhoe, 2002; Denman et al., 2007). Deter- bon degradation in Eckernförde Bay sediments in the upper mining the natural and anthropogenic sources of methane 30 cm b.s.f., followed by methanogenesis in deeper sediment is one of the major goals for oceanic, terrestrial, and atmo- layers where sulfate is depleted ( 30 cm b.s.f.; Whiticar, spheric scientists to be able to predict further impacts on the 2002; Treude et al., 2005a; Martens et al., 1998; Fig. 1). This world’s climate. The ocean is considered to be a modest nat- methanogenesis below the sulfate–methane transition zone ural source for (Wuebbles and Hayhoe, (SMTZ) can be intense and often leads to methane oversat- 2002; Reeburgh, 2007; EPA, 2010). However, research is still uration in the porewater below 50 cm of sediment depth, re- sparse on the origin of the observed oceanic methane, which sulting in gas bubble formation (Abegg and Anderson, 1997; automatically leads to uncertainties in current ocean flux es- Whiticar, 2002; Thießen et al., 2006). Thus, methane is trans- timations (Bange et al., 1994; Naqvi et al., 2010; Bakker et ported from the methanogenic zone (> 30 cm b.s.f.) to the sur- al., 2014). face sediment by both molecular diffusion and advection via Within the marine environment, the coastal areas (includ- rising gas bubbles (Wever et al., 1998; Treude et al., 2005a). ing estuaries and shelf regions) are considered the major Although upward-diffusing methane is mostly retained by source for atmospheric methane, contributing up to 75 % to the anaerobic oxidation of methane (AOM; Treude et al., the global ocean methane production (Bange et al., 1994). 2005a), a major part is reaching the sediment–water interface The majority of coastal methane is produced during micro- through gas bubble transport (Treude et al., 2005a; Jackson et bial methanogenesis in the sediment, with probably only a al., 1998), resulting in a supersaturation of the water column minor part originating from methane production within the with respect to atmospheric methane concentrations (Bange water column (Bakker et al., 2014). However, knowledge et al., 2010). The time series station Boknis Eck in the Eck- on the magnitude, seasonality, and environmental controls of ernförde Bay is a known site of methane emissions into the benthic methanogenesis is still limited.

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Figure 1. Overview of processes relevant for benthic methane production, consumption, and emission in the Eckernförde Bay. The thickness of arrows for emissions and coupling between surface processes indicates the strength of methane supply. Note that this figure combines existing knowledge with results from the present study. See discussion for more details. atmosphere throughout the year due to this supersaturation SRZ methanogenesis (1) is controlled by environmental pa- of the water column (Bange et al., 2010). rameters, (2) shows seasonal variability, and/or (3) is based The source for benthic and water column methane was on noncompetitive substrates with a special focus on methy- seen in methanogenesis below the SMTZ ( 30 cm b.s.f.; lotrophic methanogens. Whiticar, 2002); however, the coexistence of sulfate re- duction and methanogenesis has been postulated (Whiticar, 2002; Treude et al., 2005a). Still, the magnitude and envi- 2 Material and methods ronmental controls of SRZ methanogenesis are poorly un- derstood, even though SRZ methanogenesis may make a 2.1 Study site measurable contribution to benthic methane emissions given Samples were taken at the time series station Boknis Eck the short diffusion distance to the sediment–water interface (BE; 54◦31.15 0N, 10◦02.18 0E; http://www.bokniseck.de) lo- (Knittel and Boetius, 2009). The production of methane cated at the entrance of Eckernförde Bay in the southwestern within the sulfate reduction zone of Eckernförde Bay sedi- Baltic Sea with a water depth of about 28 m (map of sampling ments could further explain the peaks in methane oxidation site can be found in Hansen et al., 1999). From mid-March observed in top sediment layers, which was previously at- until mid-September the water column is strongly stratified tributed to methane transported to the sediment surface via due to the inflow of saltier North Sea water and warmer and rising gas bubbles (Treude et al., 2005a). fresher surface water (Bange et al., 2011). Organic matter In the present study, we investigated sediments from degradation in the deep layers causes pronounced within (< 30 cm b.s.f., on a seasonal basis) and below the sul- (March–September) or even anoxia (August–September; fate reduction zone ( 30 cm b.s.f., on one occasion) and the Smetacek, 1985; Smetacek et al., 1984). The source of or- water column (on a seasonal basis) at the time series station ganic material is phytoplankton blooms that occur regularly Boknis Eck in Eckernförde Bay to validate the existence of in spring (February–March) and fall (September–November) SRZ methanogenesis and its potential contribution to ben- and are followed by the pronounced sedimentation of organic thic methane emissions. Water column parameters like oxy- matter (Bange et al., 2011). To a lesser extent, phytoplank- gen, temperature, and salinity together with porewater geo- ton blooms and sedimentation are also observed during the chemistry and benthic methanogenesis were measured over summer months (July–August; Smetacek et al., 1984). Sed- the course of 2 years. In addition to seasonal rate measure- iments at BE are generally classified as soft, fine-grained ments, inhibition and stimulation experiments, stable isotope muds (< 40 µm) with a carbon content of 3 to 5 wt % (Balzer probing, and molecular analysis were carried out to find out if et al., 1986). The bulk of organic matter in Eckernförde Bay www.biogeosciences.net/15/137/2018/ Biogeosciences, 15, 137–157, 2018 140 J. Maltby et al.: Microbial methanogenesis in the sulfate-reducing zone sediments originates from marine plankton and macroalgal followed by storage at room temperature until further treat- sources (Orsi et al., 1996), and its degradation leads to the ment. production of free methane gas (Wever and Fiedler, 1995; Concentrations of dissolved methane (CH4) were deter- Abegg and Anderson, 1997; Wever et al., 1998). The oxy- mined by headspace gas chromatography as described in gen penetration depth is limited to the upper few millimeters Bange et al. (2010). Calibration for CH4 was done by using when bottom waters are oxic (Preisler et al., 2007). Reduc- a two-point calibration with known methane concentrations ing conditions within the sulfate reduction zone lead to a dark before the measurement of headspace gas samples, resulting gray or black sediment color with a strong hydrogen in an error of < 5 %. odor in the upper meter of the sediment and a dark olive- Water samples for chlorophyll concentration were taken green color in the deeper sediment layers (> 1 m; Abegg and by transferring the complete water volume (from 25 m wa- Anderson, 1997). ter of depth) from one water sampler into a 4.5 L Nalgene bottle, from which approximately 0.7–1 L (depending on 2.2 Water column and sediment sampling the plankton content) were filtrated back in the GEOMAR laboratory using a GF/F filter (Whatman; 25 mm diameter, 8 µM pores size). Dissolved chlorophyll a concentrations Sampling was done on a seasonal basis during the years were determined using the fluorometric method described by 2013 and 2014. One-day field trips with either RV Alkor Welschmeyer (1994) with an error of < 10 %. (cruise no. AL410), RV Littorina, or RV Polarfuchs were conducted in March, June, and September of each year. In 2.4 Sediment porewater geochemistry 2013, additional sampling was conducted in November. In each sampling month, water profiles of temperature, salinity, Porewater was extracted from sediment within 24 h after core and oxygen concentration (optical sensor RINKO III; detec- retrieval using nitrogen (N ) pre-flushed rhizons (0.2 µm; tion limit = 2 µM) were measured with a CTD (Hydro-Bios). 2 Rhizosphere Research Products; Seeberg-Elverfeldt et al., In addition, water samples for methane concentration mea- 2005). In MUC cores, rhizons were inserted into the sedi- surements were taken at 25 m of water depth with a Niskin ment in 2 cm intervals through pre-drilled holes in the core bottle (4 L each) rosette attached to the CTD (Table 1). Com- liner. In the gravity core, rhizons were inserted into the sedi- plementary samples for water column chlorophyll were taken ment in 30 cm intervals directly after retrieval. at 25 m of water depth with the CTD rosette within the Extracted porewater from MUC and gravity cores was im- same months during standardized monthly sampling cruises mediately analyzed for sulfide using standardized photomet- to Boknis Eck organized by GEOMAR. ric methods (Grasshoff et al., 1999). Sediment cores were taken with a miniature multi- Sulfate concentrations were determined using ion chro- corer (MUC; K.U.M. Kiel), holding four core liners matography (Metrohm 761). Analytical precision was < 1 % (length = 60 cm, diameter = 10 cm) at once. The cores had based on repeated analysis of IAPSO seawater standards (di- an average length of ∼ 30 cm and were stored at 10 ◦C in lution series) with an absolute detection limit of 1 µM cor- a cold room (GEOMAR) until further processing (normally responding to a detection limit of 30 µM for the undiluted within 1–3 days after sampling). sample. In September 2013, a gravity core was taken in addi- For analysis of dissolved inorganic carbon (DIC), 1.8 mL tion to the MUC cores. The gravity core was equipped with of porewater was transferred into a 2 mL glass vial, fixed with an inner plastic bag (polyethylene; diameter: 13 cm). Af- 10 µL saturated HgCL solution, and crimp sealed. DIC con- ter core recovery (330 cm total length), the polyethylene 2 centration was determined as CO with a multi N/C 2100 bag was cut open at 12 different sampling depths, result- 2 analyzer (Analytik Jena) following the manufacturer instruc- ing in intervals of 30 cm, and sampled directly onboard for tions. Therefore, the sample was acidified with phosphoric sediment porewater geochemistry (see Sect. 2.4), sediment acid and the outgassing CO was measured. The detection methane (see Sect. 2.5), sediment solid-phase geochemistry 2 limit was 20 µM with a precision of 2–3 %. (see Sect. 2.6), and microbial rate measurements for hy- drogenotrophic methanogenesis as described in Sect. 2.8. 2.5 Sediment methane concentrations

2.3 Water column parameters In March 2013, June 2013, and March 2014, one MUC core was sliced in 1 cm intervals until 6 cm b.s.f. followed by 2 cm In each sampling month, water samples for methane con- intervals until the end of the core. In the other sampling centration measurements were taken at 25 m of water depth months, the MUC core was sliced in 1 cm intervals until in triplicates. Therefore, three 25 mL glass vials were filled 6 cm b.s.f. followed by 2 cm intervals until 10 cm b.s.f. and bubble free directly after CTD rosette recovery and closed 5 cm intervals until the end of the core. with butyl rubber stoppers. Biological activity in samples Per sediment depth (in MUC and gravity cores), 2 cm−3 was stopped by adding saturated mercury chloride solution of sediment were transferred into a 10 mL glass vial contain-

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Table 1. Sampling months with bottom water (∼ 2 m above the seafloor) temperature (Temp.), dissolved oxygen (O2), and dissolved methane (CH4) concentration.

Sampling month Date Instrument Temp. O2 CH4 Type of (◦C) (µM) (nM) analysis March 2013 13.03.2013 CTD 3 340 30 WC MUC All June 2013 27.06.2013 CTD 6 94 125 WC MUC All September 2013 25.09.2013 CTD 10 bdl 262∗ WC MUC All GC GC-All November 2013 08.11.2013 CTD 12 163 13 WC MUC All March 2014 13.03.2014 CTD 4 209 41∗ WC MUC All June 2014 08.06.2014 CTD 7 47 61 WC MUC All September 2014 17.09.2014 CTD 13 bdl 234 WC MUC All

MUC: multicorer, GC: gravity corer, CTD: CTD rosette, bdl: below detection limit (5 µM), All: methane gas analysis, porewater analysis, sediment geochemistry, net methanogenesis analysis, hydrogenotrophic methanogenesis analysis, GC-All: analysis for gravity cores including methane gas analysis, porewater analysis, sediment geochemistry, hydrogenotrophic methanogenesis analysis, WC: water column analyses including methane analysis, chlorophyll analysis. ∗ Concentrations from the regular monthly Boknis Eck sampling cruises on 24 September 2013 and 5 March 2014 (www.bokniseck.de). ing 5 mL NaOH (2.5 %) for the determination of sediment 2.7 Sediment methanogenesis methane concentration per volume of sediment. The vial was quickly closed with a butyl septum, crimp sealed, and shaken 2.7.1 Methanogenesis in MUC cores thoroughly. The vials were stored upside down at room tem- perature until measurement via gas chromatography. There- In each sampling month, three MUC cores were sliced fore, 100 µL of headspace was removed from the gas vials in 1 cm intervals until 6 cm b.s.f., in 2 cm intervals until and injected into a Shimadzu gas chromatograph (GC-2014) 10 cm b.s.f., and in 5 cm intervals until the bottom of the core. equipped with a packed Haysep-D column and a flame ion- Every sediment layer was transferred to a separate beaker ization detector. The column temperature was 80 ◦C and the and quickly homogenized before subsampling. The exposure −1 time with air, i.e., oxygen, was kept to a minimum. Sedi- helium flow was set to 12 mL min . CH4 concentrations ment layers were then sampled for the determination of net were calibrated against CH4 standards (Scotty gases). The detection limit was 0.1 ppm with a precision of 2 %. methanogenesis (defined as the sum of total methane produc- tion and consumption, including all available methanogenic substrates in the sediment), hydrogenotrophic methanogen- esis (methanogenesis based on the substrates CO2 and H2), 2.6 Sediment solid-phase geochemistry and potential methanogenesis (methanogenesis at ideal con- ditions, i.e., no lack of nutrients) as described in the follow- ing sections. Following the sampling for CH4, the same cores described under Sect. 2.5 were used for the determination of the sedi- Net methanogenesis ment solid-phase geochemistry, i.e., porosity, particulate or- ganic carbon (POC), and particulate organic nitrogen (PON). Net methanogenesis was determined with sediment slurry The sediment porosity of each sampled sediment section experiments by measuring the headspace methane concen- was determined by the weight difference of 5 cm−3 of wet tration over time. Per sediment layer, triplicates of 5 cm−3 of sediment after freeze-drying for 24 h. Dried sediment sam- sediment were transferred into N2-flushed sterile glass vials ples were then used for analysis of particulate organic carbon (30 mL) and mixed with 5 mL of filtered bottom water. The (POC) and particulate organic nitrogen (PON) with a Carlo slurry was repeatedly flushed with N2 to remove residual Erba element analyzer (NA 1500). The detection limit for C methane and to ensure complete anoxia. Slurries were incu- and N analysis was < 0.1 dry weight percent (%) with a pre- bated in the dark at in situ temperature, which varied for each cision of < 2 %. sampling date (Table 1). Headspace samples (0.1 mL) were www.biogeosciences.net/15/137/2018/ Biogeosciences, 15, 137–157, 2018 142 J. Maltby et al.: Microbial methanogenesis in the sulfate-reducing zone taken out every 3–4 days over a time period of 4 weeks and Molybdate was used as an enzymatic inhibitor for sulfate re- analyzed on a Shimadzu GC-2104 gas chromatograph (see duction (Oremland and Capone, 1988) and BES was used as Sect. 2.5). Net methanogenesis rates were determined by the an inhibitor for methanogenic (Hoehler et al., 1994). linear increase in the methane concentration over time (min- Methanol is a known noncompetitive substrate, which is used imum of six time points; see also Fig. S1 in the Supplement). by methanogens but not by sulfate reducers (Oremland and Polcin, 1982), and thus it is suitable to examine noncompet- Hydrogenotrophic methanogenesis itive methanogenesis. Treatments were incubated similar to net methanogenesis (see the previous paragraph about net To determine if hydrogenotrophic methanogenesis, i.e., methanogenesis) by incubating sediment slurries at the re- methanogenesis based on the competitive substrate H2, is spective in situ temperature (Table 1) in the dark for a time present in the sulfate-reducing zone, radioactive sodium bi- period of 4 weeks. Headspace samples (0.1 mL) were taken 14 carbonate (NaH CO3) was added to the sediment. every 3–5 days over a time period of 4 weeks and potential Per sediment layer, sediment was sampled in triplicates methanogenesis rates were determined by the linear increase with glass tubes (5 mL) that were closed with butyl rubber in methane concentration over time (minimum of six time stoppers on both ends according to Treude et al. (2005b). points). 14 Through the stopper, NaH CO3 (dissolved in water, injec- tion volume 6 µL, activity 222 kBq, specific activity = 1.85– Potential methylotrophic methanogenesis from methanol 2.22 GBq mmol−1) was injected into each sample and in- using stable isotope probing cubated for 3 days in the dark at in situ temperature (Ta- ble 1). To stop bacterial activity, sediment was transferred One additional experiment was conducted with sediments into 50 mL glass vials filled with 20 mL of sodium hydrox- from September 2014 by adding 13C-labeled methanol to in- ide (2.5 % w/w), closed quickly with rubber stoppers, and vestigate the production of 13C-labeled methane. Three cores shaken thoroughly. Five controls were produced from vari- were stored at 1 ◦C after the September 2014 cruise until ous sediment depths by injecting the radiotracer directly into further processing ∼ 3.5 months later. The low storage tem- the NaOH with sediment. perature together with the expected oxygen depletion in the The production of 14C-methane was determined with the enclosed supernatant water after the retrieval of the cores slightly modified method by Treude et al. (2005b) used for likely led to slowed anaerobic microbial activity during stor- the determination of the anaerobic oxidation of methane. The age time and preserved the sediments for potential methano- method was identical, except no unlabeled methane was de- genesis measurements. termined by using gas chromatography. Instead, DIC values Sediment cores were sliced in 2 cm intervals and the up- were used to calculate hydrogenotrophic methane produc- per 0–2 cm b.s.f. sediment layer of all three cores was com- tion. bined in a beaker and homogenized. Then, sediment slurries were prepared by mixing 5 cm−3 of sediment with 5 mL of Potential methanogenesis in manipulated experiments artificial seawater medium in N2-flushed, sterile glass vials (30 mL). After this, methanol was added to the slurry with To examine the interaction between sulfate reduction and a final concentration of 10 mM (see also the previous para- methanogenesis, inhibition and stimulation experiments graph about potential methanogenesis in manipulated exper- were carried out. Therefore, every other sediment layer was iments). Methanol was enriched with 13C-labeled methanol sampled resulting in the following examined six sediment in a ratio of 1 : 1000 between 13C-labeled (99.9 % 13C) and layers: 0–1, 2–3, 4–5, 6–8, 10–15, and 20–25 cm. From each non-labeled methanol mostly consisting of 12C (manufac- layer, sediment slurries were prepared by mixing 5 mL of turer: Roth). In total, 54 vials were prepared for nine dif- sediment in a 1 : 1 ratio with an adapted artificial seawater ferent sampling time points during a total incubation time of ◦ medium (salinity 24; Widdel and Bak, 1992) in N2-flushed, 37 days. All vials were incubated at 13 C (in situ tempera- sterile glass vials before further manipulations. ture in September 2014) in the dark. At each sampling point, In total, four different treatments, each in triplicates, six vials were stopped: one set of triplicates was used for were prepared per depth: (1) with sulfate addition (17 mM), headspace methane and carbon dioxide determination and a (2) with sulfate (17 mM) and molybdate (22 mM) addi- second set of triplicates was used for porewater analysis. tion, (3) with sulfate (17 mM) and 2-bromoethanesulfonate Headspace methane and carbon dioxide concentrations (BES; 60 mM) addition, and (4) with sulfate (17 mM) and (volume 100 µL) were determined on a Shimadzu gas chro- methanol (10 mM) addition. From here on, the following matograph (GC-2014) equipped with a packed Haysep-D names are used to describe the different treatments, re- column, a flame ionization detector, and a methanizer. The spectively: (1) control treatment, (2) molybdate treatment, methanizer (reduced nickel) reduces carbon dioxide with (3) BES treatment, and (4) methanol treatment. Control treat- hydrogen to methane at a temperature of 400 ◦C. The col- ments feature the natural sulfate concentrations occurring in umn temperature was 80 ◦C and the helium flow was set sediments of the sulfate reduction zone at the sampling site. to 12 mL min−1. Methane concentrations (including reduced

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◦ CO2) were calibrated against methane standards (Scotty frozen at −20 C. DNA was extracted from ∼ 500 mg of gases). The detection limit was 0.1 ppm with a precision of sediment using the FastDNA® SPIN Kit for Soil (Biomed- 2 %. ical). The quantitative real-time polymerase chain reaction Analyses of the 13C / 12C ratios of methane and car- (qPCR) technique using TaqMan probes and TaqMan chem- bon dioxide were conducted after headspace concentration istry (Life Technologies) was used for the detection of measurements by using a continuous-flow combustion gas methanogens on a ViiA7 qPCR machine (Life Technolo- chromatograph (Trace Ultra; Thermo Scientific), which was gies). Primer and probe sets as originally published by Yu et coupled to an isotope ratio mass spectrometer (MAT253; al. (2005) were applied to quantify the orders Methanobac- Thermo Scientific). The isotope ratios of methane and car- teriales, Methanosarcinales, and Methanomicrobiales along bon dioxide given in the common delta notation (δ13C in with the two families Methanosarcinaceae and Methanosae- permill) are reported relative to Vienna Pee Dee Belemnite taceae within the order Methanosarcinales. In addition, a uni- (VPDB) standard. Isotope precision was ±0.5 ‰ when mea- versal primer set for the detection of the Archaea was suring near the detection limit of 10 ppm. used (Yu et al., 2005). For porewater analysis of methanol concentration and iso- Absolute quantification of the 16S rDNA from the groups tope composition, each sediment slurry of the triplicates was mentioned above was performed with standard dilution se- transferred into argon-flushed 15 mL centrifuge tubes and ries. The standard concentration reached from 108 to 101 centrifuged for 6 min at 4500 rpm. Then 1 mL of filtered copies per µL. Quantification of the standards and samples (0.2 µm) porewater was transferred into N2-flushed 2 mL was performed in duplicates. Reaction was performed in a glass vials for methanol analysis, crimp sealed, and immedi- final volume of 12.5 µL containing 0.5 µL of each primer ately frozen at −20 ◦C. Methanol concentrations and isotope (10 pmol µL−1; MWG), 0.25 µL of the respective probe −1 composition were determined via high-performance liquid (10 pmol µL ; Life Technologies), 4 µL of H2O (Roth), chromatography–ion ratio mass spectrometry (HPLC-IRMS; 6.25 µL of TaqMan Universal Master Mix II (Life Technolo- Thermo Fisher Scientific) at the MPI Marburg. The detection gies), and 1 µL of sample or standard. Cycling conditions limit was 50 µM with a precision of 0.3 ‰. started with an initial denaturation and activation step for 10 min at 95 ◦C followed by 45 cycles of 95 ◦C for 15 s, 56 ◦C 2.7.2 Methanogenesis in the gravity core for 30 s, and 60 ◦C for 60 s. Non-template controls were run in duplicates with water instead of DNA for all primer and Ex situ hydrogenotrophic methanogenesis was determined probe sets and remained without any detectable signal after in a gravity core taken in September 2013. The pathway is 45 cycles. thought to be the main methanogenic pathway in the sedi- ment below the SMTZ in Eckernförde Bay (Whiticar, 2002). Hydrogenotrophic methanogenesis was determined using ra- 2.9 Statistical analysis 14 dioactive sodium bicarbonate (NaH CO3). At every sam- pled sediment depth (12 depths in 30 cm intervals), tripli- cate glass tubes (5 mL) were inserted directly into the sedi- To determine the possible environmental control parame- ment. Tubes were filled bubble free with sediment and closed ters of SRZ methanogenesis, a principal component analysis with butyl rubber stoppers on both ends according to Treude (PCA) was applied according to the approach described in et al. (2005). The methods following sampling were identi- Gier et al. (2016). Prior to PCA, the dataset was transformed cal to those described in the previous paragraph about hy- into ranks to ensure the same data dimensions. drogenotrophic methanogenesis. In total, two PCAs were conducted. The first PCA was used to test the relation of parameters in the surface sed- 2.8 Molecular analysis iment (integrated methanogenesis (0–5 cm, mmol m−2 d−1), POC content (average value from 0–5 cm b.s.f., wt %), C / N During the non-labeled methanol treatment of the 0– (average value from 0–5 cm b.s.f., molar) and the bottom wa- 1 cm b.s.f. horizon from the September 2014 sampling (see ter (25 m of water depth) oxygen (µM), temperature (◦C), also the previous paragraph about potential methanogenesis salinity (PSU), chlorophyll (µg L−1), and methane (nM). The in manipulated experiments), additional samples were pre- second PCA was applied on depth profiles of sediment SRZ pared to detect and quantify the presence of methanogens methanogenesis (nmol cm−3 d−1), sediment depth (cm), sed- in the sediment. Therefore, an additional 15 vials were pre- iment POC content (wt %), sediment C / N ratio (molar), and pared with the addition of methanol as described in the pre- sampling month (one value per depth profile at a specific vious paragraph about potential methanogenesis in manipu- month, the later in the year the higher the value). lated experiments for five different time points (day 1 (= t0), For each PCA, biplots were produced to view data from day 8, day 16, day 22, and day 36) and stopped at each different angles and to graphically determine a potential pos- time point by transferring sediment from the triplicate slur- itive, negative, or zero correlation between methanogenesis ries into whirl-paks (Nasco), which then were immediately rates and the tested variables. www.biogeosciences.net/15/137/2018/ Biogeosciences, 15, 137–157, 2018 144 J. Maltby et al.: Microbial methanogenesis in the sulfate-reducing zone

Figure 2. Parameters measured in the water column and sediment in the Eckernförde Bay in each sampling month in the year 2013. Net methanogenesis (MG) and hydrogenotrophic (hydr.) methanogenesis rates are shown in triplicates with mean (solid line).

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3 Results tions at a shallower sediment depth late in the year. The mag- nitudes of methane concentrations were similar in the respec- 3.1 Water column parameters tive months of 2013 and 2014. In all sampling months, the sulfide concentration increased From March 2013 to September 2014, the water column with sediment depth (Figs. 2 and 3). Similar to methane, sul- had pronounced temporal and spatial variability in temper- fide profiles revealed higher sulfide concentrations at a shal- ature, salinity, and oxygen (Figs. 2 and 3). In 2013, the tem- lower sediment depth together with higher peak concentra- perature of the upper water column increased from March tions over the course of the sampled months in each sampling (1 ◦C) to September (16 ◦C), but decreased again in Novem- year. Accordingly, November 2013 (10.5 mM at 15 cm b.s.f.) ber (11 ◦C). The temperature of the lower water column in- and September 2014 (2.8 mM at 15 cm b.s.f.) revealed the creased from March 2013 (2 ◦C) to November 2013 (12 ◦C). highest sulfide concentrations. September 2014 was the only In 2014, the lowest temperatures of the upper and lower wa- sampling month showing a pronounced decrease in sulfide ter column were reached in March (4 ◦C). Warmer tempera- concentration from 15 to 21 cm b.s.f. of over 50 %. tures of the upper water column were observed in June and DIC concentrations increased with increasing sediment September (around 17 ◦C), while the lower water column depth in all sampling months. Concomitant with the high- peaked in September (13 ◦C). est sulfide concentrations, the highest DIC concentration was Salinity increased over time during 2013, showing the detected in November 2013 (26 mM at 27 cm b.s.f.). At the highest salinity of the upper and lower water column in surface, DIC concentrations ranged between 2 and 3 mM in November (18 and 23 PSU, respectively). In 2014, the salin- all sampling months. In June of both years, DIC concentra- ity of the upper water column was highest in March and tions were lowest at the deepest sampled depth compared to September (both 17 PSU) and lowest in June (13 PSU). The the other sampling months (16 mM in 2013, 13 mM in 2014). salinity of the lower water column increased from March In all sampling months, POC profiles scattered around 2014 (21 PSU) to September 2014 (25 PSU). 5 ± 0.9 wt % with depth. Only in November 2013, June 2014, In both years, June and September showed the most pro- and September 2014 did POC content exceed 5 wt % in the nounced vertical gradient of temperature and salinity, featur- upper 0–1 cm b.s.f. (5.9, 5.2, and 5.3 wt %, respectively) with ing a pycnocline at around ∼ 14 m of water depth. the highest POC content in November 2013. Also in Novem- Summer stratification was also seen in the O2 profiles, ber 2013, the surface C / N ratio (0–1 cm b.s.f.) of the partic- which showed O2 depleted conditions (O2 < 150 µM) in the ulate organic matter was the lowest of all sampling months lower water column from June to September in both years, (8.6). In general, the C / N ratio increased with depth in both reaching concentrations below 1–2 µM (detection limit of years with values around 9 at the surface and values around CTD sensor) in September of both years (Figs. 2 and 3). 10–11 at the deepest sampled sediment depths. The water column was completely ventilated, i.e., homog- enized, in March of both years with O2 concentrations of 3.3 Sediment geochemistry in gravity cores 300–400 µM down to the seafloor at about 28 m. Results from sediment porewater and solid-phase geochem- 3.2 Sediment geochemistry in MUC cores istry in the gravity core from September 2013 are shown in Fig. 4. Please note that the sediment depth of the grav- Sediment porewater and solid-phase geochemistry results for ity core was corrected by comparing the sulfate concentra- the years 2013 and 2014 are shown in Figs. 2 and 3, respec- tions at 0 cm b.s.f. in the gravity core with the correspond- tively. ing sulfate concentration and depth in the MUC core from Sulfate concentrations at the sediment surface ranged be- September 2013 (Fig. 2). The soft surface sediment is often tween 15 and 20 mM. The concentration decreased with lost during the gravity coring procedure. Through this correc- depth in all sampling months but was never fully depleted tion, the topmost layer of the gravity core was set at a depth until the bottom of the core (18–29 cm b.s.f.; between 2 and of 14 cm b.s.f. 7 mM sulfate). November 2013 showed the strongest de- Porewater sulfate concentration in the gravity core de- crease from ∼ 20 mM at the top to ∼ 2 mM at the bottom creased with depth (i.e., below 0.1 mM at 107 cm b.s.f.) and of the core (27 cm b.s.f.). stayed below 0.1 mM until 324 cm b.s.f. Sulfate increased Opposite to sulfate, the methane concentration increased slightly (1.9 mM) at the bottom of the core (345 cm b.s.f.). with sediment depth in all sampling months (Figs. 2 and 3). In concert with sulfate, methane, sulfide, DIC, POC, and Over the course of a year (i.e., March to November in 2013 C / N profiles also showed distinct alteration in the profile and March to September in 2014), the maximum methane at 345 cm b.s.f. (see below, Fig. 4). As fluid seepage has concentration increased, reaching the highest concentration not been observed at the Boknis Eck station (Schlüter et in November 2013 (∼ 1 mM at 26 cm b.s.f.) and September al., 2000), these alterations could either indicate a change in 2014 (0.2 mM at 23 cm b.s.f.). Simultaneously, methane pro- sediment properties or result from a sampling artifact from files became steeper, revealing higher methane concentra- the penetration of seawater through the core catcher into the www.biogeosciences.net/15/137/2018/ Biogeosciences, 15, 137–157, 2018 146 J. Maltby et al.: Microbial methanogenesis in the sulfate-reducing zone

deepest sediment layer. The latter process is, however, not ex- pected to considerably affect sediment solid-phase properties (POC and C / N), and we therefore dismissed this hypothesis. The methane concentration increased steeply with depth, reaching a maximum of 4.8 mM at 76 cm b.s.f. The concen- tration stayed around 4.7 mM until 262 cm b.s.f. followed by a slight decrease until 324 cm b.s.f. (2.8 mM). From 324 to 345 cm b.s.f. methane increased again (3.4 mM). Both sulfide and DIC concentrations increased with depth, showing a maximum at 45 (∼ 5 mM) and 345 cm b.s.f. (∼ 1 mM), respectively. While sulfide decreased after 45 cm b.s.f. to a minimum of ∼ 300 µM at 324 cm b.s.f., it slightly increased again to ∼ 1 mM at 345 cm b.s.f. In ac- cordance, DIC concentrations showed a distinct decrease be- tween 324 and 345 cm b.s.f. (from 45 to 39 mM). While POC contents varied around 5 wt % throughout the core, the C / N ratio slightly increased with depth, revealing the lowest ratio at the surface (∼ 3) and the highest ratio at the bottom of the core (∼ 13). However, both POC and C / N showed a distinct increase from 324 to 345 cm b.s.f.

3.4 Methanogenesis activity in MUC cores

3.4.1 Net methanogenesis

Net methanogenesis activity (calculated by the linear in- crease of methane over time; see Fig. S1) was detected throughout the cores in all sampling months (Figs. 2 and 3). Activity measured in MUC cores increased over the course of the year in 2013 and 2014 (that is, March to November in 2013 and March to September in 2014) with lower rates mostly < 0.1 in March and higher rates > 0.2 nmol cm−3 d−1 in November 2013 and September 2014. In general, Novem- ber 2013 revealed the highest net methanogenesis rates (1.3 nmol cm−3 d−1 at 1–2 cm b.s.f.). Peak rates were de- tected at the sediment surface (0–1 cm b.s.f.) in all sam- pling months except for September 2013 when the maxi- mum rates were situated between 10 and 15 cm b.s.f. In addi- tion to the surface peaks, net methanogenesis showed sub- surface (= below 1 until 30 cm b.s.f.) maxima in all sam- pling months, but with alternating depths (between 10 and 25 cm b.s.f.). A comparison of the integrated net methanogenesis rates (0–25 cm b.s.f.) revealed the highest rates in September and November 2013 (0.09 and 0.08 mmol m−2 d−1, respectively) and the lowest rates in March 2014 (0.01 mmol m−2 d−1; Fig. 5). A trend of increasing areal net methanogenesis rates from March to September was observed in both years.

3.4.2 Hydrogenotrophic methanogenesis

Figure 3. Parameters measured in the water column and sediment Hydrogenotrophic methanogenesis activity determined by in the Eckernförde Bay in each sampling month in the year 2014. 14C-bicarbonate incubations of MUC cores is shown in Net methanogenesis (MG) and hydrogenotrophic (hydr.) methano- Figs. 2 and 3. In 2013, maximum activity ranged between genesis rates are shown in triplicates with mean (solid line). 0.01 and 0.2 nmol cm−3 d−1, while in 2014 maxima ranged

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Figure 4. Parameters measured in the sediment gravity core taken in the Eckernförde Bay in September 2013. Hydrogenotrophic (hydr.) methanogenesis rates are shown in triplicates with mean (solid line).

3.4.3 Potential methanogenesis in manipulated experiments

Potential methanogenesis rates in manipulated experiments included either the addition of inhibitors (molybdate for the inhibition of sulfate reduction or BES for the inhibition of methanogenesis) or the addition of a noncompetitive sub- strate (methanol). Control treatments were run with neither the addition of inhibitors nor the addition of methanol. Controls. Potential methanogenesis activity in the control treatments was below 0.5 nmol cm−3 d−1 from March 2014 Figure 5. Integrated net methanogenesis (MG) rates (determined to September 2014 (Fig. 6). Only in November 2013 did con- −3 −1 by net methane production) and hydrogenotrophic MG rates (de- trol rates exceed 0.5 nmol cm d below 6 cm b.s.f. While termined by radiotracer incubation) in the surface sediments (0– rates increased with depth in November 2013 and June 2014, 25 cm b.s.f.) of Eckernförde Bay for different sampled time points. they decreased with depth in the other two sampling months. Molybdate. Peak potential methanogenesis rates in the molybdate treatments were found in the uppermost sedi- ment interval (0–1 cm b.s.f.) in almost every sampling month only between 0.01 and 0.05 nmol cm−3 d−1. In comparison, with rates being 3–30 times higher compared to the con- maximum hydrogenotrophic methanogenesis was up to 2 or- trol treatments (< 0.5 nmol cm−3 d−1). In November 2013, ders of magnitude lower compared to net methanogenesis. potential methanogenesis showed two maxima (0–1 and Only in March 2013 did both activities reach a similar range. 10–15 cm b.s.f.). The highest measured rates were found in Overall, hydrogenotrophic methanogenesis increased with September 2014 (∼ 6 nmol cm−3 d−1) followed by Novem- depth in March, September, and November 2013 and in ber 2013 (∼ 5 nmol cm−3 d−1). March, June, and September 2014. In June 2013, activity de- BES. Profiles of potential methanogenesis in the BES creased with depth, showing the highest rates in the upper treatments were similar to the controls mostly in the lower 0–5 cm b.s.f. and the lowest at the deepest sampled depth. range < 0.5 nmol cm−3 d−1. Only in November 2013 did Concomitant with integrated net methanogenesis, rates exceed 0.5 nmol cm−3 d−1. Rates increased with depth integrated hydrogenotrophic methanogenesis rates (0– in all sampling months, except for September 2014, when 25 cm b.s.f.) were high in September 2013, with slightly the highest rates were found at the sediment surface (0– higher rates in March 2013 (Fig. 5). The lowest areal rates 1 cm b.s.f.). of hydrogenotrophic methanogenesis were seen in June of Methanol. In all sampling months, potential rates in the both years. methanol treatments were 3 orders of magnitude higher com- Hydrogenotrophic methanogenesis activity in the grav- pared to the control treatments (< 0.5 nmol cm−3 d−1). Ex- ity core is shown in Fig. 4. The highest activity cept for November 2013, potential methanogenesis rates (∼ 0.7 nmol cm−3 d−1) was measured at 45 and 138 cm b.s.f. in the methanol treatments were highest in the upper 0– followed by a decrease with increasing sediment depth 5 cm b.s.f. and decreased with depth. In November 2013, the reaching 0.01 nmol cm−3 d−1 at the deepest sampled depth highest rates were detected at the deepest sampled depth (20– (345 cm b.s.f.). 25 cm b.s.f.). www.biogeosciences.net/15/137/2018/ Biogeosciences, 15, 137–157, 2018 148 J. Maltby et al.: Microbial methanogenesis in the sulfate-reducing zone

Figure 6. Potential methanogenesis rates versus sediment depth in sediment sampled in November 2013, March 2014, June 2014, and September 2014. Presented are four different types of incubations (treatments): control (blue symbols) describes the treatment with sediment plus artificial seawater containing natural salinity (24 PSU) and sulfate concentrations (17 mM), molybdate (green symbols) is the treatment with the addition of molybdate (22 mM), BES (purple symbols) is the treatment with 60 mM BES addition, and methanol (red symbols) is the treatment with the addition of 10 mM of methanol. Shown are triplicates per depth interval and the mean as a solid line. Please note the different x axis for the methanol treatment (red).

3.4.4 Potential methanogenesis followed by Please note that the majority of CO2 was dissolved in the 13 C-methanol labeling porewater, and thus the CO2 content in the headspace does not show the total CO2 abundance in the system. CO2 in the Total methanol concentrations (labeled and unlabeled) in the headspace was enriched with 13C during the first 2 weeks sediment decreased sharply in the first 2 weeks from ∼ 8 mM (from −16.2 to −7.3 ‰) but then stayed around −11 ‰ un- at day 1 to 0.5 mM at day 13 (Fig. 7). At day 17, methanol til the end of the incubation. was below the detection limit. In the first 2 weeks, residual methanol was enriched with 13C, reaching ∼ 200 ‰ at day 3.5 Molecular analysis of benthic methanogens 13. Over the same time period, the methane content in the headspace increased from 2 ppmv at day 1 to ∼ 66 000 ppmv In September 2014, additional samples were run during the at day 17 and stayed around that value until the end of the methanol treatment (see Sect. 2.7.) for the detection of ben- total incubation time (until day 37; Fig. 7). The carbon iso- thic methanogens via qPCR. The qPCR results are shown in 13 topic signature of methane (δ CCH4 ) showed a clear en- Fig. 8. For a better comparison, the microbial abundances are richment of the heavier isotope 13C (Table 3) from day 9 plotted together with the sediment methane concentrations to 17 (no methane was detectable at day 1). After day 17, from the methanol treatment, from which the rate calculation 13 δ CCH4 stayed around 13 ‰ until the end of the incuba- for the methanol-methanogenesis at 0–1 cm b.s.f. was done tion. The content of CO2 in the headspace increased from (shown in Fig. 6). ∼ 8900 ppmv at day 1 to ∼ 29 000 ppmv at day 20 and stayed Sediment methane concentrations increased over time, re- around 30 000 ppmv until the end of the incubation (Fig. 7). vealing a slow increase in the first ∼ 10 days followed by a

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Figure 8. Sediment methane concentrations (with sediment from the 0–1 cm b.s.f. in September 2014) over time in the treatment Figure 7. Development of headspace gas content and isotope com- with the addition of methanol (10 mM) are shown above. Shown position of methane (CH4) and carbon dioxide (CO2) as well as are triplicate values per measurement. DNA copies of Archaea, porewater methanol (CH3OH) concentration and isotope composi- Methanosarcinales, and Methanosarcinaceae are shown below in tion during the 13C-labeling experiment (with sediment from the duplicates per measurement. Please note the secondary y axis for 0–2 cm b.s.f. horizon in September 2014) with the addition of 13C- Methanosarcinales and Methanosarcinaceae. More data are avail- enriched methanol (13C:12C = 1:1000). (a) Concentrations of pore- able for methane (determined in the gas headspace) than from DNA water methanol (CH3OH) and headspace content of methane (CH4) samples (taken from the sediment) as sample volume for molecular and carbon dioxide (CO2) over time. (b) Isotope composition of analyzes was limited. porewater CH3OH, headspace CH4, and headspace CO2 over time. Shown are means (from triplicates) with standard deviation. 3.6 Statistical analysis steep increase between day 13 and day 20 and ending in a The PCA of integrated SRZ methanogenesis (0–5 cm b.s.f.; stationary phase. Fig. 10) showed a positive correlation with bottom water A similar increase was seen in the abundance of to- temperature (Fig. 10a), bottom water salinity (Fig. 10a), bot- tal and methanogenic Archaea. Total Archaea abundances tom water methane (Fig. 10b), surface sediment POC con- increased sharply in the second week of the incubation, tent (0–5 cm b.s.f.; Fig. 10c), and surface sediment C / N (0– reaching a maximum at day 16 (∼ 5000 × 106 copies g−1), 5 cm b.s.f.; Fig. 10b). A negative correlation was found with and stayed around 3000 × 106–4000 × 106 copies g−1 over bottom water oxygen concentration (Fig. 10b). No correla- the course of the incubation. Similarly, methanogenic ar- tion was found with bottom water chlorophyll. chaea, namely the order Methanosarcinales and within this The PCA of methanogenesis depth profiles showed pos- order the family Methanosarcinaceae, showed a sharp in- itive correlations with sediment depth (Fig. 11a) and C / N crease in the first 2 weeks as well with the highest abun- (Fig. 11b), and it showed negative correlations with POC dances at day 16 (∼ 6 × 108 and ∼ 1 × 106 copies g−1, re- (Fig. 11a). spectively). Until the end of the incubation, the abundances of Methanosarcinales and Methanosarcinaceae decreased to about one-third of their maximum abundances (∼ 2 × 108 4 Discussion and ∼ 0.4 × 106 copies g−1, respectively). 4.1 Methanogenesis in the sulfate-reducing zone

On the basis of the results presented in Figs. 2 and 3, it is evident that methanogenesis and sulfate reduction were con- currently active in the sulfate reduction zone (0–30 cm b.s.f.) www.biogeosciences.net/15/137/2018/ Biogeosciences, 15, 137–157, 2018 150 J. Maltby et al.: Microbial methanogenesis in the sulfate-reducing zone

3. The addition of BES did not result in the inhibition of methanogenesis, indicating the presence of unconven- tional methanogenic groups using noncompetitive sub- strates (Fig. 7). The unsuccessful inhibition by BES can be explained by either incomplete inhibition or the fact that the methanogens were insensitive to BES (Hoehler et al., 1994; Smith and Mah, 1981; Santoro and Konisky, 1987). The BES concentration applied in the present study (60 mM) has been shown to result in the successful inhibition of methanogens in previ- ous studies (Hoehler et al., 1994). Therefore, the pres- Figure 9. Temporal development of integrated net surface methano- ence of methanogens that are insensitive to BES is more genesis (0–5 cm b.s.f.) in the sediment and chlorophyll (green) likely. The insensitivity to BES in methanogens is ex- and methane concentrations (orange) in the bottom water (25 m). plained by heritable changes in BES permeability or the Methanogenesis (MG) rates and methane concentrations are shown formation of BES-resistant enzymes (Smith and Mah, in means (from triplicates) with standard deviation. 1981; Santoro and Konisky, 1987). Such BES resis- tance was found in mutants (Smith and Mah, 1981; Santoro and Konisky, 1987). This genus at Boknis Eck. Even though sulfate reduction activity was was successfully detected in our samples (for more de- not directly determined, the decrease in sulfate concentra- tails see point 5) and is known for mediating the methy- tions with a concomitant increase in sulfide within the up- lotrophic pathway (Keltjens and Vogels, 1993), support- per 30 cm b.s.f. clearly indicated its presence (Figs. 2 and 3). ing our hypothesis on the utilization of noncompetitive Several previous studies confirmed the high activity of sul- substrates by methanogens. fate reduction in the surface sediment of Eckernförde Bay, revealing rates up to 100–10 000 nmol cm−3 d−1 in the upper 4. The addition of methanol to sulfate-rich sediments in- 25 cm b.s.f. (Treude et al., 2005a; Bertics et al., 2013; Dale et creased methanogenesis rates by up to 3 orders of mag- al., 2013). The microbial of organic matter was nitude, confirming the potential of the methanogenic probably high in the organic-rich sediments of Eckernförde community to utilize noncompetitive substrates, espe- Bay (POC contents of around 5 %; Figs. 2 and 3), providing cially in the 0–5 cm b.s.f. sediment horizon (Fig. 6). high substrate availability and variety for methanogenesis. At this sediment depth either the availability of non- The results of this study further identified methylotrophy competitive substrates, including methanol, was high- to be a potentially important noncompetitive methanogenic est (derived from fresh organic matter), or the usage pathway in the sulfate-reducing zone. The pathway utilizes of noncompetitive substrates was increased due to the alternative substrates, such as methanol, to bypass compe- high competitive situation as sulfate reduction is most tition with sulfate reducers for H2 and acetate. The poten- active in the 0–5 cm b.s.f. layer (Treude et al., 2005a; tial for methylotrophic methanogenesis within the sulfate- Bertics et al., 2013). It should be noted that even though reducing zone was supported by the following observations. methanogenesis rates were calculated assuming a linear increase in methane concentration over the entire incu- 1. Hydrogenotrophic methanogenesis was up to 2 orders bation to make a better comparison between different of magnitude lower compared to net methanogenesis, treatments, the methanol treatments generally showed resulting in insufficient rates to explain the observed a delayed response in methane development (Figs. 8, net methanogenesis in the upper 0–30 cm b.s.f. (Figs. 2 S2). We suggest that this delayed response was a reflec- and 3). This finding points towards the presence of alter- tion of cell growth by methanogens utilizing the surplus native methanogenic processes in the sulfate reduction methanol. We are therefore unable to decipher whether zone, such as methylotrophic methanogenesis. methanol plays a major role as a substrate in the Eck- ernförde Bay sediments compared to possible alterna- 2. Methanogenesis increased when sulfate reduction was tives, as its concentration is relatively low in the natural inhibited by molybdate, confirming the inhibitory effect setting (∼ 1 µM between 0 and 25 cm b.s.f., June 2014 of sulfate reduction on methanogenesis with competi- sampling; Zhuang, unpublished data). It is conceivable tive substrates (H and acetate; Oremland and Polcin, 2 that other noncompetitive substrates, such as methy- 1982; King et al., 1983; Fig. 6). Consequently, the usage lated sulfides (e.g., dimethyl sulfide or ), of noncompetitive substrates was preferred in the sulfate are more relevant for the support of SRZ methanogene- reduction zone (especially in the upper 0–1 cm b.s.f.; sis. Fig. 6). Accordingly, hydrogenotrophic methanogenesis increased at depths at which sulfate was depleted and 5. Methylotrophic methanogens of the order thus the competitive situation was relieved (Fig. 4). Methanosarcinales were detected in the methanol treat-

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Figure 10. Principal component analysis (PCA) from three different angles of integrated surface methanogenesis (0–5 cm b.s.f.) and surface particulate organic carbon averaged over 0–5 cm b.s.f. (surface sediment POC), surface C / N ratio averaged over 0–5 cm b.s.f. (surface sediment C / N), bottom water salinity, bottom water temperature (T ), bottom water methane (CH4), bottom water oxygen (O2), and bottom water chlorophyll. Data were transformed into ranks before analysis. (a) Correlation biplot of principal components 1 and 2, (b) correlation biplot of principal components 1 and 3, and (c) correlation biplot of principal components 2 and 3. Correlation biplots are shown in a multidimensional space with parameters shown as green lines and samples shown as black dots. Parameters pointing in the same direction are positively related; parameters pointing in the opposite direction are negatively related.

ment (Fig. 8), confirming the presence of methanogens 6. Stable isotope probing revealed highly 13C-enriched that utilize noncompetitive substrates in the natural methane produced from 13C-labeled methanol, further environment (Boone et al., 1993; Fig. 8). The delay confirming the potential of the methanogenic commu- in the growth of Methanosarcinales moreover hints nity to utilize noncompetitive substrates (Fig. 7). The towards the predominant usage of noncompetitive production of both methane and CO2 from methanol substrates other than methanol (see also point 4). has been shown previously in different strains of methy- www.biogeosciences.net/15/137/2018/ Biogeosciences, 15, 137–157, 2018 152 J. Maltby et al.: Microbial methanogenesis in the sulfate-reducing zone

lotrophic methanogens (Penger et al., 2012). The fast conversion of methanol to methane and CO2 (methanol was consumed completely in 17 days) hints towards the presence of methylotrophic methanogens (e.g., mem- bers of the family Methanosarcinaceae, which is known for the methylotrophic pathway; Keltjens and Vogels, 1993). Please note, however, that the storage of the cores (3.5 months) prior to sampling could have led to shifts in the microbial community and thus might not reflect the in situ conditions of the original microbial community in September 2014. The delay in methane production also seen in the stable isotope experiment was, however, only slightly different (methane devel- oped earlier between day 8 and 12; data not shown) from the non-labeled methanol treatment (between day 10 and 16; Fig. S2), which leads us to the assumption that the storage time at 1 ◦C did not dramatically affect the methanogen community. Similar to a previous study with arctic sediments, the addition of substrates had no stimulatory effect on the rate of methanogenesis or on the methanogen community structure at low tempera- tures (5 ◦C; Blake et al., 2015).

4.2 Environmental control of methanogenesis in the sulfate reduction zone Figure 11. Principal component analysis (PCA) from two different SRZ methanogenesis in Eckernförde Bay sediments showed angles of net methanogenesis depth profiles and sampling month variations throughout the sampling period, which may be in- (Month), sediment depth, and depth profiles of particulate organic fluenced by variable environmental factors such as tempera- carbon (POC) and C / N ratio (C / N). Data were transformed into ture, salinity, oxygen, and organic carbon. In the following, ranks before analysis. (a) Correlation biplot of principal compo- we will discuss the potential impact of those factors on the nents 1 and 2 and (b) correlation biplot of principal components 1 magnitude and distribution of SRZ methanogenesis. and 3. Correlation biplots are shown in a multidimensional space with parameters shown as green lines and samples shown as black 4.2.1 Temperature dots. Parameters pointing in the same direction are positively re- lated; parameters pointing in the opposite direction are negatively During the sampling period, bottom water temperatures in- related. creased over the course of the year from late winter (March, 3–4 ◦C) to autumn (November, 12 ◦C; Figs. 2 and 3). The PCA revealed a positive correlation between bottom wa- and Martens, 1981; Crill and Martens, 1983; Martens and ter temperature and integrated SRZ methanogenesis (0– Klump, 1984). 5 cm b.s.f.). A temperature experiment conducted with sed- iment from ∼ 75 cm b.s.f. in September 2014 within a par- 4.2.2 Salinity and oxygen allel study revealed a mesophilic temperature optimum of methanogenesis (20 ◦C; data not shown). Whether methano- From March 2013 to November 2013 and from March 2014 genesis in the sulfate reduction zone (0–30 cm) has the to September 2014, salinity increased in the bottom-near wa- same physiology remains speculative. However, AOM organ- ter (25 m) from 19 to 23 and from 22 to 25 PSU (Figs. 2 isms, which are closely related to methanogens (Knittel and and 3), respectively, due the pronounced summer stratifica- Boetius, 2009), studied in the sulfate reduction zone from tion in the water column between saline North Sea water the same site were confirmed to have a mesophilic physi- and less saline Baltic Sea water (Bange et al., 2011). The ology, too (Treude et al., 2005a). The sum of these aspects PCA detected a positive correlation between integrated SRZ leads us to the conceivable conclusion that SRZ methanogen- methanogenesis (0–5 cm b.s.f.) and salinity in the bottom- esis activity in the Eckernförde Bay is positively impacted near water (Fig. 10a). This correlation can hardly be ex- by temperature increases. Such a correlation between ben- plained by salinity alone, as methanogens feature a broad thic methanogenesis and temperature has been found in sev- salinity range from freshwater to hypersaline (Zinder, 1993). eral previous studies from different environments (Sansone It is more likely that salinity serves as an indicator of wa-

Biogeosciences, 15, 137–157, 2018 www.biogeosciences.net/15/137/2018/ J. Maltby et al.: Microbial methanogenesis in the sulfate-reducing zone 153 ter column stratification, which is often correlated with low a positive correlation between integrated SRZ methanogen- O2 concentrations in the Eckernförde Bay (Fig. S3, Bange et esis rates and bottom-near water methane concentrations al., 2011; Bertics et al., 2013). Methanogenesis is sensitive (Fig. 10b) when viewed over all investigated months. How- to O2 (Oremland, 1988; Zinder, 1993), and hence conditions ever, no correlation was found between bottom water chloro- might be more favorable during hypoxic or anoxic events, phyll and integrated SRZ methanogenesis rates (Fig. 10). particularly in the sediment closest to the sediment–water in- As seen in Fig. 9, bottom-near high chlorophyll concentra- terface, but potentially also in deeper sediment layers due to tions did not coincide with high bottom-near methane con- the absence of bioturbating and bioirrigating infauna (Dale centration in June–September 2014. We explain this result et al., 2013; Bertics et al., 2013), which could introduce O2 by a time lag between in the water col- beyond diffusive transport. Accordingly, the PCA revealed a umn and the export of the produced organic material to the negative correlation between O2 concentration close to the seafloor, which was probably even more delayed during strat- seafloor and SRZ methanogenesis. ification. Such a delay was observed in a previous study (Bange et al., 2010), revealing an enhanced water methane 4.2.3 Particulate organic carbon concentration close to the seafloor approximately 1 month after the chlorophyll maximum. The C / N ratio (averaged The supply of particulate organic carbon (POC) is one of over 0–5 cm b.s.f.) also showed no correlation with integrated the most important factors controlling benthic heterotrophic methanogenesis from the same depth layer (0–5 cm b.s.f.), processes, as it determines substrate availability and variety which is surprising as we expected that a higher C / N ra- (Jørgensen, 2006). In Eckernförde Bay, the organic mate- tio indicative of less labile organic carbon would have a rial reaching the seafloor originates mainly from phytoplank- negative effect on noncompetitive methanogenesis. However, ton blooms in spring, summer, and autumn (Bange et al., methanogens are not able to directly use most of the labile or- 2011). It has been estimated that > 50 % in spring (February– ganic matter due to their inability to process large molecules March), < 25 % in summer (July–August), and > 75 % in au- (more than two C–C bondings; Zinder, 1993). Methanogens tumn (September–October) of these blooms is reaching the are dependent on other microbial groups to degrade large or- seafloor (Smetacek et al., 1984), resulting in an overall high ganic compounds (e.g., amino acids) for them (Zinder, 1993). organic carbon content of the sediment (5 wt %), which leads Because of this substrate speciation and dependence, a de- to high benthic microbial degradation rates including sul- lay between the sedimentation of fresh, labile organic matter fate reduction and methanogenesis (Whiticar, 2002; Treude and the increase in methanogenesis can be expected, which et al., 2005a; Bertics et al., 2013). Previous studies revealed would not be captured by the applied PCA. that high organic matter availability can relieve competi- tion between sulfate reducers and methanogens in sulfate- 4.2.5 Effect of POC and C / N ratio over 0–30 cm b.s.f. containing marine sediments (Oremland et al., 1982; Holmer and Kristensen, 1994; Treude et al., 2009; Maltby et al., In the PCA for the sediment profiles from the sulfate reduc- 2016). tion zone (0–30 cm b.s.f.), POC showed a negative correla- To determine the effect of POC concentration and C / N ra- tion with methanogenesis and sediment depth, while C / N tio (the latter as a negative indicator for the freshness of POC) ratio showed a positive correlation with methanogenesis and on SRZ methanogenesis, two PCAs were conducted with sediment depth (Fig. 11). Given that POC remained basi- (a) the focus on the upper 0–5 cm b.s.f., which is directly in- cally unchanged over the top 30 cm b.s.f. with the exception fluenced by freshly sedimented organic material from the wa- of the topmost sediment layer, its negative correlation with ter column (Fig. 10), and (b) the focus on the depth profiles methanogenesis is probably solely explained by the increase throughout the sediment cores (up to 30 cm b.s.f.; Fig. 11). in methanogenesis with sediment depth and can therefore be excluded as a major controlling factor. As sulfate in this zone 4.2.4 Effect of POC and C / N ratio in the upper was likely never depleted to levels that critically limit sul- 0–5 cm b.s.f. fate reduction (lowest concentration 1300 µM; compare with Treude et al., 2014), we do not expect a significant change in For the upper 0–5 cm b.s.f. in the sediment, a positive corre- the competition between methanogens and sulfate reducers. lation was found between SRZ methanogenesis (integrated) It is therefore more likely that the progressive degradation and POC content (averaged; Fig. 10c), indicating that POC of labile POC into dissolvable methanogenic substrates over content is an important controlling factor for methanogen- depth and time had a positive impact on methanogenesis. The esis in this layer. In support, the highest bottom-near wa- C / N ratio indicates such a trend as the labile fraction of POC ter chlorophyll concentrations coincided with the highest decreased with depth. bottom-near water methane concentrations and high inte- grated SRZ methanogenesis (0–5 cm b.s.f.) in September 2013, probably as a result of the sedimentation of the sum- mer phytoplankton bloom (Fig. 9). Indeed, the PCA revealed www.biogeosciences.net/15/137/2018/ Biogeosciences, 15, 137–157, 2018 154 J. Maltby et al.: Microbial methanogenesis in the sulfate-reducing zone

4.3 Relevance of methanogenesis in the sulfate the order of 1.6 mmol m−2 d−1 (1.5 mmol m−2 d−1 AOM + reduction zone of Eckernförde Bay sediments 0.09 mmol m−2 d−1 net SRZ methanogenesis). Even though the contribution of SRZ methanogenesis to AOM above the SMTZ remains speculative, it leads to the as- The time series station Boknis Eck in Eckernförde Bay sumption that SRZ methanogenesis could play a much bigger is known for being a methane source to the atmosphere role for benthic carbon cycling in the Eckernförde Bay than throughout the year due to supersaturated waters, which previously thought. Whether SRZ methanogenesis at Eckern- result from significant benthic methanogenesis and emis- förde Bay has the potential for the direct emission of methane sion (Bange et al., 2010). The benthic methane formation is into the water column goes beyond the scope of this study thought to take place mainly in sediments below the SMTZ and should be tested in the future. (Treude et al., 2005a; Whiticar, 2002). In the present study, we show that SRZ methanogenesis within the sulfate zone is present despite sulfate concen- 5 Summary trations > 1 mM, a limit above which methanogenesis has The present study demonstrated that methanogenesis and sul- been thought to be negligible (Alperin et al., 1994; Hoehler fate reduction were concurrently active within the sulfate- et al., 1994; Burdige, 2006), and could thus contribute to reducing zone in sediments at Boknis Eck (Eckernförde Bay, benthic methane emissions. In support of this hypothesis, a SW Baltic Sea). The observed methanogenesis was proba- high dissolved methane concentration in the water column bly based on noncompetitive substrates due to the competi- occurred with concomitantly high SRZ methanogenesis ac- tion with sulfate reducers for the substrates H and acetate. tivity (Fig. 9). However, whether the observed water col- 2 Accordingly, members of the family Methanosarcinaceae, umn methane originated from SRZ methanogenesis, from which are known for methylotrophic methanogenesis, were gas ebullition caused by methanogenesis below the SMTZ, found in the sulfate reduction zone of the sediments and or a mixture of both remains speculative. are likely to be responsible for the observed methanogene- How much of the methane produced in the surface sed- sis with the potential use of noncompetitive substrates such iment is ultimately emitted into the water column depends as methanol, , or methylated sulfides. on the rate of methane consumption, i.e., the aerobic and Potential environmental factors controlling SRZ methano- anaerobic oxidation of methane in the sediment (Knittel and genesis are POC content, C / N ratio, oxygen, and tempera- Boetius, 2009; Fig. 1). In organic-rich sediments, such as ture, resulting in the highest methanogenesis activity during in the present study, the oxygenated sediment layer is of- the warm, stratified, and hypoxic months after the late sum- ten only millimeters thick due to the high O demand of mi- 2 mer phytoplankton blooms. croorganisms during organic matter degradation (Jørgensen, This study provides new insights into the presence and 2006; Preisler et al., 2007). Thus, the anaerobic oxidation seasonality of SRZ methanogenesis in coastal sediments and of methane (AOM) might play a more important role for was able to demonstrate that the process could play an im- methane consumption in the studied Eckernförde Bay sed- portant role for the methane budget and carbon cycling of iments. In an earlier study from this site, AOM activity Eckernförde Bay sediments, for example by directly fueling was detected throughout the top 0–25 cm b.s.f., which in- AOM above the SMTZ. cluded zones that were well above the actual SMTZ (Treude et al., 2005a). But the authors concluded that methane oxidation was completely fueled by methanogenesis from Data availability. Research data for the present study below sulfate penetration, as integrated AOM rates (0.8– can be accessed via the public data repository PANGEA −2 −1 1.5 mmol m d ) were in the same range as the predicted (https://doi.org/10.1594/PANGAEA.873185, Maltby et al., 2017). methane flux (0.66–1.88 mmol m−2 d−1) into the SMTZ. Together with the dataset presented here we postulate that − − AOM above the SMTZ (0.8 mmol m 2 d 1; Treude et al., The Supplement related to this article is available online 2005a) could be partially or entirely fueled by SRZ methano- at https://doi.org/10.5194/bg-15-137-2018-supplement. genesis. A similar close coupling between methane oxida- tion and methanogenesis in the absence of definite methane profiles was recently proposed from isotopic labeling exper- iments with sediments from the sulfate reduction zone of Author contributions. JM and TT designed the experiments. JM the nearby Aarhus Bay in Denmark (Xiao et al., 2017). It carried out all experiments. HWB coordinated measurements of is therefore likely that such a cryptic methane cycling also water column methane and chlorophyll. CRL and MAF conducted occurs in the sulfate reduction zone of sediments in the Eck- molecular analysis. MS coordinated 13C-isotope measurements. JM ernförde Bay. If, in an extreme scenario, SRZ methanogen- prepared the paper with contributions from all coauthors. esis represented the only methane source for AOM above the SMTZ, then maximum SRZ methanogenesis could be on

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Competing interests. The authors declare that they have no conflict Bange, H. W., Hansen, H. P., Malien, F., Laß, K., Karstensen, J., of interest. Petereit, C., Friedrichs, G., and Dale, A.: Boknis Eck Time Se- ries Station (SW Baltic Sea): Measurements from 1957 to 2010, LOICZ-Affiliated Activities, Inprint 20, 16–22, 2011. Acknowledgements. We thank the captain and crew of RV Alkor, Bertics, V. J., Löscher, C. R., Salonen, I., Dale, A. W., Gier, J., RV Littorina, and RV Polarfuchs for field assistance. We thank Schmitz, R. A., and Treude, T.: Occurrence of benthic micro- Gabriele Schüssler, Fynn Wulff, Peggy Wefers, Asmus Pe- bial nitrogen fixation coupled to sulfate reduction in the season- tersen, Maik Lange, and Florian Evers for field and laboratory ally hypoxic Eckernförde Bay, Baltic Sea, Biogeosciences, 10, assistance. For the geochemical analysis we want to thank Bet- 1243–1258, https://doi.org/10.5194/bg-10-1243-2013, 2013. tina Domeyer, Anke Bleyer, Ulrike Lomnitz, Regina Suhrberg, Blake, L. I., Tveit, A., Øvreås, L., Head, I. M., and Gray, N. D.: and Verena Thoenissen. We thank Frank Malien, Xiao Ma, Response of Methanogens in Arctic Sediments to Temperature Annette Kock, and Tina Baustian for the O2, CH4, and chlorophyll and Methanogenic Substrate Availability, Planet. Space Sci., 10, measurements from the regular monthly Boknis Eck sampling 1–18, 2015. cruises. Further, we thank Ralf Conrad and Peter Claus at the MPI Boone, D. R., Whitman, W. B., and Rouvière, P.: Diversity and Tax- Marburg for the 13C-methanol measurements. This study received onomy of Methanogens, in: Methanogenesis, edited by: Ferry, J. financial support through the Cluster of Excellence “The Future G., Springer US, 35–81, 1993. Ocean” funded by the German Research Foundation through Buckley, D. H., Baumgartner, L. 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